Methods of using an electrochemical biosensor

ABSTRACT

According to one embodiment of the present invention, an electrochemical sensor ( 10 ) for detecting the concentration of analyte in a fluid test sample is disclosed. The sensor ( 10 ) includes a counter electrode having a high-resistance portion for use in detecting whether a predetermined amount of sample has been received by the test sensor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/590,834, titled “Electrochemical Biosensor,” and filed on Aug. 24,2006, now allowed, which is a U.S. national stage of InternationalApplication No. PCT/US2005/04226, titled “Electrochemical Biosensor,”and filed Feb. 4, 2005, which claims priority to Application No.60/542,364, titled “Electrochemical Biosensor,” and filed on Feb. 6,2004, each of which is incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention is generally related to an electrochemicalbiosensor for use in the quantification of an analyte in a liquid sampleand, more particularly, to a system for detecting an insufficient sampleamount in an electrochemical biosensor.

BACKGROUND OF THE INVENTION

Medical conditions such as diabetes require a person afflicted with thecondition to regularly self-monitor that person's blood-glucoseconcentration level. The purpose of monitoring the blood glucoseconcentration level is to determine the person's blood glucoseconcentration level and then to take corrective action, based uponwhether the level is too high or too low, to bring the level back withina normal range. The failure to take corrective action can have seriousmedical implications for that person.

One method of monitoring a person's blood glucose level is with aportable testing device. The portable nature of these devices enablesusers to conveniently test their blood glucose levels wherever they maybe. One type of device utilizes an electrochemical biosensor to harvestthe blood sample and to analyze the blood sample. The electrochemicalbiosensor includes a reagent designed to react with glucose in the bloodto create an oxidation current at electrodes disposed within theelectrochemical biosensor—this current is indicative of the user's bloodglucose concentration level.

A predetermined amount of reagent is included within an electrochemicalbiosensor, and is designed to react with a predetermined sample volume.If a less-than required sample volume is harvested by theelectrochemical biosensor—a condition referred to as beingunder-filled—an erroneous measurement may result. Becauseelectrochemical biosensors are commonly used in a self-testingenvironment, there exists an increased chance that an inappropriateamount of sample may be collected. Further, because the sample volumesare very small (typically less than about 10 μl) it is difficult for auser to visually determine whether an appropriate amount of sample hasbeen harvested for analysis. Thus, there exists a need for anelectrochemical biosensor that reliably detects and alerts a user to theoccurrence of an under-filled condition.

SUMMARY OF THE INVENTION

According to one embodiment of the present invention, an electrochemicalsensor for detecting the concentration of analyte in a fluid test sampleis disclosed. The sensor includes a counter electrode having ahigh-resistance portion for use in detecting whether a predeterminedamount of sample has been received by the test sensor.

According to another embodiment of the present invention, a method forevaluating whether an electrochemical test sensor is properly filled isdisclosed. The test sensor includes a working electrode coupled to afirst lead and a counter electrode coupled to a second lead. The counterelectrode includes a high-resistance portion and a low-resistanceportion. The test sensor includes a reagent disposed on the workingelectrode that is adapted to react with an analyte in a fluid sample forproducing an electrochemical reaction indicative of the concentration ofthe analyte in the fluid sample. The method comprises applying a voltageprofile across the first and second leads, measuring the current profileat the first and second leads in response to the applied voltageprofile, and generating an under-filled error signal when the measuredcurrent profile does not have a predetermined profile.

The above summary of the present invention is not intended to representeach embodiment, or every aspect, of the present invention. Additionalfeatures and benefits of the present invention are apparent from thedetailed description, figures, and embodiments set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an exploded view of an electrochemical biosensor according toone embodiment of the present invention.

FIG. 2 a is an oversized top view of an electrode pattern of theelectrochemical bio sensor of FIG. 1.

FIG. 2 b is a circuitry schematic of the electrochemical biosensor ofFIG. 2 a when the electrochemical biosensor is partially filled withliquid sample.

FIG. 2 c is a circuitry schematic of the electrochemical biosensor ofFIG. 2 a when the electrochemical biosensor is appropriately filled withliquid sample.

FIG. 3 a is a plot of the voltage profile applied to the test sensor ofFIG. 1 according to one embodiment of the present invention.

FIGS. 3 b and 3 c are plots of the current profile of the test sensor inresponse to the voltage profile of FIG. 3 a in an under-filled conditionand an appropriately-filled condition, respectively.

FIG. 4 a is a plot of the voltage profile applied to the test sensor ofFIG. 1 according to another embodiment of the present invention.

FIGS. 4 b and 4 c are plots of the current profile of the test sensor inresponse to the voltage profile of FIG. 4 a in an under-filled conditionand an appropriately-filled condition, respectively.

While the invention is susceptible to various modifications andalternative forms, specific embodiments are shown by way of example inthe drawings and are described in detail herein. It should beunderstood, however, that the invention is not intended to be limited tothe particular forms disclosed.

DETAILED DESCRIPTION OF THE ILLUSTRATED EMBODIMENTS

Turning to the drawings and initially to FIG. 1, the construction of anelectrochemical sensor 10 is shown according to one embodiment of thepresent invention. The sensor 10 comprises an insulating base 12 uponwhich is printed in sequence (typically by screen printing techniques)an electrical conductor pattern including first and second leads 14 a,b,an electrode pattern including a working electrode 16, a counterelectrode, an insulating (dielectric) layer 20 including an opening 22and a channel 25, and a reaction layer 24. The counter electrodeincludes a low-resistance counter electrode branch 18 (LRC electrode)and a high-resistance counter electrode branch 19 (HRC electrode).

The reaction layer 24 includes a reagent for converting an analyte ofinterest (e.g., glucose) in a fluid test sample (e.g., blood) into achemical species that is electrochemically measurable, in terms of theelectrical current it produces, by the components of the electrodepattern. The reagent of the reaction layer 24 typically contains anenzyme such as, for example, glucose oxidase, which reacts with theanalyte and with an electron acceptor such as a ferricyanide salt toproduce an electrochemically measurable species that can be detected bythe electrode pattern 16,18,19. The reaction layer 24 comprises apolymer, an enzyme, and an electron acceptor. The reaction layer 24 alsoincludes additional ingredients such as a buffer and a surfactant insome embodiments of the present invention. The reaction layer 24 isdisposed over the opening 22 and channel 25 in the insulating layer 20.Thus, the portion of the reaction layer 24 exposed to the electrodepattern 16,18,19 is defined by an opening 22 and a channel 25 in theinsulating layer 20. The working electrode 16 is electrically coupled tothe first lead 14 a, and the LRC electrode 18 and HRC electrode 19 areelectrically coupled to a second lead 14 b.

The reaction layer 24 covers only the working electrode 16, covers theworking electrode 16 and the LRC electrode 18, or covers the workingelectrode 16, the LRC electrode 18, and the HRC electrode 19 inalternative embodiments of the present invention. When the reactionlayer 24 covers only the working electrode 16, an electroactive materialis present on the LRC electrode 18 to allow it to function as a counterelectrode as is well known in the art.

The sensor 10 includes a lid 30 having a concave portion 32 that forms acapillary channel when mated with the insulating layer 20 for moving theliquid sample from an inlet 34 into the test sensor 10. The downstreamend of the capillary channel includes one or more openings 36 forventing the capillary channel—the fluid sample flows from the inlet 34into the sensor 10 toward the opening 36. In use, the sensor 10 collectsa fluid sample (e.g., a blood sample from a patient's finger) bybringing the capillary channel inlet 34 into contact with the fluidsample.

Referring to FIG. 2 a, the working electrode 16 and LRC electrode 18 areconfigured in a manner such that the LRC electrode 18 is locateddownstream (in terms of the direction of fluid sample flow along theflow path) from the working electrode 16. This configuration offers theadvantage of requiring the test fluid to completely cover the workingelectrode 16 in order to contact the LRC electrode 18. However, the HRCelectrode 19, which is coupled to the LRC electrode 18 via a resistor40, is positioned upstream from the working electrode 16. According toone embodiment of the present invention, the resistor 40 has aresistance of about 50 kΩ to about 500 kΩ. In other embodiments, theresistance of the resistor 40 ranges between about 250 kΩ to about 350kΩ. In yet another embodiment, the resistor 40 has a resistance of about300 kΩ. The resistor 40 may be screen-printed on the insulating base 12in a manner similar to the working electrode 16, the LRC electrode 18,the HRC electrode 19, and the leads 14 a,b. Generally, as describedbelow, the resistor 40 is used in detecting an under-filled condition inthe test sensor 10, which can result in an inaccurate measurement of theanalyte of interest in the fluid sample.

Referring to FIG. 2 b, the working electrode 16 and HRC electrode 19form the circuit illustrated if the sensor 10 is under-filled (i.e., theLRC electrode 18 in FIG. 2 a is not covered by the fluid sample). Inthis situation, the sensor current passes through the resistor 40. Thus,the potential V₂ between the working electrode 16 and the HRC electrode19 is about the difference between the potential V₁ applied to thesensor leads 14 a,b and the voltage drop V_(r) across the resistor 40,assuming a negligible resistance along the electrode/lead pattern.

Referring to FIG. 2 c, the working electrode 16 and the LRC electrode 18form the illustrated circuit if the sensor 10 is appropriately filled(i.e., the LRC electrode 18 in FIG. 2 a is covered by the liquidsample). In this situation, the resistor 40 is electrically bypassed inthe circuitry. Thus, the potential V₂ between the working electrode 16and the LRC electrode 18 is substantially the same as the potential V₁applied to the leads 14 a,b of the sensor 10, assuming a negligibleresistance along the electrode/lead pattern. The current measured in thesensor 10 is a result of diffusion of electro-active species to theelectrodes and the subsequent redox reactions there. For example, at theworking electrode 16 an electron is taken from ferrocyanide, oxidizingit to ferricyanide. At the LRC electrode 18 (or at the HRC electrode 19in an under-filled situation), an electron is added to ferricyanide,reducing it to ferrocyanide. The flow of electrons in the electricalpattern connecting the two electrodes is measured and is related to theamount of ferrocyanide and hence to the amount of glucose in the sample.In normal operation, a relatively high electrical potential, V₂ in FIG.2 c, is applied between the electrodes (e.g., about 400 mV), making theoxidation and reduction reactions at the electrodes fast and depletingthe region around the working electrode 16 of the reduced mediator(e.g., ferrocyanide). Thus, the current is not constant but decays withtime as the reaction is limited by the diffusion to the electrodesurface of the reduced mediator. In general, such decaying current i canbe described according to equation (1):i=C·G·t ^(−k)  (1)In equation (1), C is a constant, G is the concentration of the analyte(e.g., glucose) in the liquid sample, t is the time elapsed since thepotential V₂ is applied, and k is a constant relating to the currentdecay profile.

If a higher electrical potential is applied, no increase in the sensorcurrent is measured, and no change to the decay with time is measuredbecause the sensor current is determined by diffusion to the electrodesurface. If a lower electrical potential (e.g., about 200 mV) is appliedbetween the electrodes, the oxidation and reduction reactions areslower, but fast enough that the sensor current remains dependent ondiffusion. Eventually at a lower voltage (e.g., less than about 200 mV),local depletion of reduced mediator does not occur and the sensorcurrent ceases to vary with time. Thus, during normal operation of thesensor 10, no change in the current decay profile occurs with time overa range of applied potentials.

The operation of the test sensor 10 with under-fill detection will bedescribed. If the sensor 10 is under-filled (i.e., less than a requisiteamount for the designed reaction) the sample only covers the HRCelectrode 19 and at least a portion of the working electrode 16. In thisunder-filled situation, the HRC electrode 19 serves as the entirecounter electrode with a high-resistance due to the resistor 40. FIG. 2b illustrates the circuit under this condition. Current flow through theresistance 40 causes a potential drop V_(r) over the resistor 40 andreduces the potential V₂ available for the electrochemical reactions. Ifthe resistance is high enough, the potential V₂ is reduced to a pointwhere the electrode surface reactions are slow and the current measuredbetween electrode leads 14 a and 14 b does not decay normally with timebut is essentially flat. This flat equilibrium current is a dynamicbalance between the sensor current and voltage drop V_(r) on theresistor. Changing the applied voltage V₁ changes this equilibriumcurrent—a lower voltage results in a lower equilibrium or steady-statecurrent and a higher voltage results in a higher current. The sensorcurrent has a “step” profile if a step-shaped voltage profile isapplied.

In the situation where the sensor 10 is appropriately filled, the samplecovers the LRC electrode 18, in addition to the HRC electrode 19, andthe working electrode 16. FIG. 2 c illustrates the circuitry under thiscondition. The branch of the circuitry between the HRC electrode 19 andthe resistor 40 to the lead 14 b is electrically bypassed by the directconnection between the LRC electrode 18 and the lead 14 b. The workingelectrode 16 and the LRC electrode 18 form a low-resistance circuit, andthe sensor current has a decay-type profile where the current is limitedby diffusion of electro-active species to the electrode surface asdescribed above.

The present invention provides an electrochemical sensor in which theelectrodes are configured so that in the event of an under-filledcondition, the result is a current response with time and/or appliedvoltage that is characteristic, and can be distinguished from theresponse of a correctly-filled sensor. Specifically, there are at leasttwo ways in which to distinguish a partially-filled sensor 10 from asensor 10 that is appropriately filled according to alternativeembodiments of the present invention. First, the sensor current of apartially-filled sensor 10 does not decay normally with time, unlike thesensor current of an appropriately-filled sensor 10. Second, the sensorcurrent of a partially-filled test sensor 10 increases with appliedvoltage due to the resistor 40, while the sensor current of anappropriately-filled sensor 10 (which bypasses the resistor) does not.

Thus, when the amount of test fluid that enters the capillary space ofthe test sensor 10 is sufficient only to cover the HRC electrode 19 andat least a portion of the working electrode 16, and when a suitablepotential is applied, the current measured across leads 14 a,b isessentially constant and not decay normally with time. Put another way,a device coupled to the leads 14 a,b senses certain characteristics ofthe sensor current over time, which are used to determine if anunder-filled error condition has occurred. This is accomplished byalgorithmically programming the device to detect the under-filledcondition by measuring the current at definite time periods after thetest fluid has electrically connected the HRC electrode 19 with theworking electrode 16, and/or after the test fluid has electricallyconnected the working electrode 16 with the LRC electrode 18.

Referring to FIGS. 3 a, 3 b, and 3 c, one method for determining whetherthe test sensor 10 is appropriately filled will be described. At timet₀, a voltage step is applied between the leads 14 a,b and held constantuntil time t₁, this period is referred to as the burn period. Next, novoltage (e.g., an open circuit) is applied during a wait period fromtime t₁ to time t₂. Finally, the voltage step is again applied during aread period from t₂ to t₄. According to one embodiment of the presentinvention, the burn, wait, and read periods are each about 2 to about 10seconds in duration. The applied step voltage is about 0.3 Volts toabout 0.4 Volts, according to one embodiment of the present invention.

An under-filled sensor 10 generates a flat sensor current profile duringthe read period as shown, for example, in FIG. 3 b. Anappropriately-filled sensor 10 generates a typical decay-type sensorcurrent profile during the read period as shown, for example, in FIG. 3c.

The decay factor, k, during the read period—from time t₂ to t₄—iscalculated from the two currents, I_(r3) and I_(r4), measured at t₃ andt₄, according to equation (2):

$\begin{matrix}{k = \frac{{\ln\left( I_{r\; 3} \right)} - {\ln\left( I_{r\; 4} \right)}}{{\ln\left( t_{4} \right)} - {\ln\left( t_{3} \right)}}} & (2)\end{matrix}$In equation (2), the decay factor, k, describes how fast the current idecays in equation (1), where C is a constant, G is the glucoseconcentration, and t is the time elapsed after the voltage is initiallyapplied. In an appropriately-filled sensor 10, k is typically betweenabout 0.30 and about 0.49, decreasing as glucose concentrationincreases. The decay factor drops to zero in under-filled conditions.Therefore, an under-filled sensor 10 is detected by checking if thedecay factor is below a pre-determined lower limit.

Referring to FIGS. 4 a, 4 b, and 4 c, another method for determiningwhether the test sensor 10 is appropriately filled will be described. Afirst voltage is applied during the burn period occurring from time t₀to time t₁, and a second higher voltage is applied until time t₂. Novoltage is applied (e.g., an open circuit) during the wait period fromtime t₂ to time t₃. Finally, a voltage is applied during the read periodfrom time t₃ to time t₅. According to one embodiment, the first voltageapplied during the burn period from time t₀ to t₁ is about 0.3 V, andthe second applied during the burn period from time t₁ to time t₂ isabout 0.6 V. During the read period, a voltage of about 0.3 V isapplied. The burn, wait, and read periods are each about 2 seconds toabout 10 seconds in length, with the first voltage of the burn periodapplied for about 25% to about 75% of the total burn period, accordingto one embodiment of the present invention.

An under-filled sensor 10 generates a step sensor current profile I_(b)during the burn period as shown, for example, in FIG. 4 b. Anappropriately-filled sensor 10 generates a decay-shaped sensor currentprofile as shown, for example, in FIG. 4 c.

The decay factor k during the burn period is calculated from the twocurrents, I_(b1) and I_(b2), measured at t₁ and t₂, respectively,according to equation (3):

$\begin{matrix}{k = \frac{{\ln\left( I_{b\; 1} \right)} - {\ln\left( I_{b\; 2} \right)}}{{\ln\left( t_{2} \right)} - {\ln\left( t_{1} \right)}}} & (3)\end{matrix}$During the burn period the decay factor is greater than about 0.2 in anappropriately-filled sensor, but drops below about −1.0 in anunder-filled condition. Thus, an under-filled condition is detected bycomparing the actual decay factor to a pre-determined lower limit duringthe burn period.

According to alternative embodiments, the two algorithms—equations (2)and (3)—for detecting an under-filled condition discussed in connectionwith FIGS. 3 a-c and 4 a-c are used jointly to determine whether anunder-filled condition has occurred. The decay factor is first evaluatedduring the burn period as described in connection with FIGS. 4 a-c. Ifno under-filled condition is determined, the decay factor is thenevaluated during the read period as described in connection with FIGS. 3a-c. If no under-filled condition is detected during the burn and readperiods, an appropriately-filled condition is deemed to have occurred.

While the invention is susceptible to various modifications andalternative forms, specific embodiments thereof are shown by way ofexample in the drawings and described in detail herein. It should beunderstood, however, that it is not intended to limit the invention tothe particular forms disclosed, but on the contrary, the intention is tocover all modifications, equivalents, and alternatives falling withinthe spirit and scope of the invention as defined by the appended claims.

What is claimed is:
 1. A method for evaluating whether anelectrochemical sensor is properly filled, the electrochemical sensorincluding a working electrode and a counter electrode, the counterelectrode including a high-resistance portion and a low-resistanceportion, the high-resistance portion including a resistor, the methodcomprising: introducing the fluid test sample to the electrochemicaltest sensor; applying a first potential between a counter electrode anda working electrode for a first predetermined time period after thefluid test sample has electrically connected the working electrode andthe counter electrode; after the first predetermined time period,applying a second potential between the counter electrode and theworking electrode for a second predetermined time period, the secondpotential having a magnitude that is greater than a magnitude of thefirst potential; measuring the current between the counter electrode andthe working electrode at a first interval during the first predeterminedtime period to obtain a first current measurement; measuring the currentbetween the counter electrode and the working electrode at a secondinterval during the second predetermined period to obtain a secondcurrent measurement; determining a first parameter based on the firstcurrent measurement and the second current measurement; comparing thefirst parameter to at least one first predetermined parameter; andnotifying a user that an insufficient quantity of fluid test sample hasbeen introduced to the electrochemical test sensor based on thecomparing the first parameter to the at least one first predeterminedparameter.
 2. The method of claim 1, wherein the first parameter isdeterminable by a calculation having the general formfirst parameter=ln(I _(t1))−ln(t ₂)/(ln(t2)−ln(t1)) where I_(t1) is thefirst current measurement at the first interval, t1, and I_(t2) is thesecond current measurement at the second interval, t2.
 3. The method ofclaim 1, wherein the second current measurement has a magnitude that isgreater than a magnitude of the first current measurement when aninsufficient quantity of fluid test sample has been introduced to theelectrochemical test sensor.
 4. The method of claim 1, furthercomprising: after the second time period, applying a third potentialbetween the counter electrode and the working electrode for a thirdpredetermined time period; measuring the current between the counterelectrode and the working electrode at a third interval during the thirdpredetermined time period to obtain a third current measurement;measuring the current between the counter electrode and the workingelectrode at a fourth interval during the third predetermined timeperiod to obtain a fourth current measurement; determining a secondparameter based on the third current measurement and the fourth currentmeasurement; comparing a second parameter to at least one secondpredetermined parameter; and notifying a user that an insufficientquantity of fluid test sample has been introduced to the electrochemicaltest sensor based on the comparing the second parameter to the at leastone second predetermined parameter.
 5. The method of claim 4, whereinthe second parameter is determinable by a calculation having the generalformsecond parameter=ln(I _(t3))−ln(I _(t4))/(ln(t4)−ln(t3)) where I_(t3) isthe third current measurement at the third interval, t3, and I_(t4) isthe fourth current measurement at the fourth interval, t4.
 6. The methodof claim 4, wherein the first predetermined time period and the secondpredetermined time period comprise a burn period, the thirdpredetermined time period comprises a read period, and no potential isapplied between the counter electrode and the working electrode for await period, the wait period being after the burn period and prior tothe read period.
 7. The method of claim 4, wherein the firstpredetermined time period and the second predetermined time periodcomprise a first pulse period, and the third predetermined time periodcomprises a second pulse period.
 8. A method for evaluating whether anelectrochemical test sensor is properly filled, the test sensorincluding a working electrode electrically coupled to a first lead and acounter electrode electrically coupled to a second lead, the counterelectrode including a low-resistance portion and a high-resistanceportion, the high-resistance portion including a resistor, the methodcomprising: applying a voltage profile across the first and secondleads; measuring the current profile at the first and second leads inresponse to the applied voltage profile; comparing the measured currentprofile to a predetermined current profile; and generating anunder-filled error signal based on the comparing of the measured currentprofile to the predetermined profile.
 9. The method of claim 8 whereinthe predetermined profile is a decay-shaped profile.
 10. The method ofclaim 8 comprising forming an electrical communication between only thehigh-resistance portion of the counter electrode and the workingelectrode when the test sensor is under-filled.
 11. The method of claim8 comprising forming an electrical communication between thelow-resistance and high-resistance portions of the counter electrode andthe working electrode when the test sensor is appropriately-filled. 12.The method of claim 8 wherein the fluid test sample comprises blood. 13.The method of claim 8 wherein the analyte comprises glucose.
 14. Amethod for evaluating whether an electrochemical sensor is properlyfilled, the test sensor including a working electrode coupled to a firstlead, and counter electrode coupled to a second lead, the counterelectrode including a high-resistance portion and a low-resistanceportion, the high-resistance portion including a resistor, the testsensor including a reagent disposed on the working electrode that isadapted to react with glucose in a blood sample for producing anelectrochemical reaction indicative of the concentration of the glucosein the blood sample, the method comprising: collecting a blood sample;applying a voltage profile across the first and second leads; measuringthe current profile at the first and second leads in response to theapplied voltage profile; and generating an under-filled error signalwhen the measured current profile is not of a predetermined shape. 15.The method of claim 14 wherein the predetermined shape is adecay-shaped.
 16. The method of claim 14 comprising forming anelectrical communication between only the high-resistance portion of thecounter electrode and the working electrode when the test sensor isunder-filled.
 17. The method of claim 14 comprising forming anelectrical communication between the low-resistance portion of thecounter electrode and the working electrode when the test sensor isappropriately-filled.
 18. The method of claim 14 wherein the reagentcomprises glucose oxidase.
 19. The method of claim 14 wherein thereagent is also disposed on the low-resistance portion of the counterelectrode.
 20. The method of claim 14, wherein the resistor is screenprinted on an insulating base of the test sensor.
 21. The method ofclaim 14, wherein the reagent is also disposed on the high-resistanceportion.
 22. The method of claim 14, wherein the test sensor includes aflow path for receiving the blood sample, the low-resistance portion ofthe counter electrode is disposed along the flow path downstream fromthe working electrode, and the high-resistance portion of the counterelectrode is disposed along the flow path upstream from the workingelectrode.
 23. A method for evaluating whether an electrochemical sensoris properly filled, the test sensor including a working electrodecoupled to a first lead, and counter electrode coupled to a second lead,the counter electrode including a high-resistance portion and alow-resistance portion, the high-resistance portion including aresistor, the method comprising: collecting a blood sample; applying afirst potential across the first and second leads for a firstpredetermined time period; after the first predetermined time period,applying a second potential across the first and second leads for asecond predetermined time period, the second potential having amagnitude that is greater than a magnitude of the first potential; andmeasuring the current at the first and second leads at least oneinterval in response to the applied first potential and the appliedsecond potential.
 24. The method of claim 23, further comprisinggenerating an under-filled error signal based on the measured current.